ABSTRACT Lymphangioleiomyomatosis (LAM) is a rare, low grade neoplasia affecting primarily women of childbearing age. LAM is characterized by the nodular proliferation of SMA and HMB-45 positive LAM cells throughout the lung, producing cystic destruction and respiratory insufficiency. Mutations in the tsc2 or tsc1 genes leading to activation of the mTOR pathway underlie the disease. LAM cells express mainly progesterone receptors (PRs) rather than estrogen receptors; however the role of PRs in the disease has not been investigated. Preliminary studies using a new, well characterized, sporadic-LAM-derived LAM cell line (LAM1) which expresses PRs suggested that the progesterone (P4)/PR axis stimulates LAM cell proliferation, extracellular matrix (ECM) proteinases expression, and pericellular proteolysis, all independently of mTOR activation, whereas mifepristone, a P4 antagonist, abolishes these effects. In a separate pilot study we found that TSC2 transfer suppresses PR expression in LAM1 cells. Additionally, we found that a LAM cell subpopulation expresses PLAP (placental alkaline phosphatase) and preliminary studies suggested a direct correlation between PR positivity and level of secreted PLAP. Finally, intratracheal delivery of LAM1 cells to ovariectomized immunodeficient female mice receiving P4 resulted in LAM-like features undistinguishable from human pulmonary LAM. Based on these observations we hypothesize that: A. the P4/PRs axis contributes to the pathogenesis of LAM by stimulating LAM cell malignant behavior and lung cystic destruction; B. PR expression in LAM results from lack of TSC2-mediated PR transcription repression. C. PLAP levels in serum are a marker for PR positive (PR+) LAM cell burden; and D. concomitant blockage of mTOR and the P4/PR axis is more effective in preventing/ameliorating LAM manifestations in an orthotopic mouse model that blockage of mTOR alone. To test these hypotheses we specifically propose: 1. A To determine the involvement of the P4/PR axis in the promotion of LAM cellular features associated with a low grade neoplastic behavior, including LAM1 cell proliferation, death, production of ECM proteolytic enzymes, etc. 1. B To determine the involvement of the P4/PR axis in the development of pulmonary LAM-like disease in an orthotopic mouse model by quantifying LAM cell burden, cystic destruction, and metastatic dissemination. 1. C To determine the effect of the P4 antagonists mifepristone and lonaprisan on LAM cell low grade neoplastic behavior in vitro and in vivo using the same read outs presented in sub-aims A and B. 2. To determine the effect of TSC2 transfer on pr transcription by LAM1 cells using qPCR, promoter reporter assays and ChIP analysis. 3. To assess whether PLAP level is a marker of PR+ LAM cell burden by determining the potential in vitro and in vivo correlation between PR+ LAM cell burden and PLAP levels in medium and serum. 4. To compare the effectivity of rapamycin alone versus rapamycin plus a P4 antagonist in the treatment of LAM-like disease in an orthotopic mouse model. The experimental protocol will be modeled on that presented in aim 1 B, with same read outs.